Levin and Bang first reported in 1964 that lysate made from the amebocytes of Limulus polyphemus (horseshoe crab) coagulated in the presence of bacterial endotoxin, and currently it has been improved to detect picogram (10.sup.-12 grams) quantities of endotoxin. Due to its extreme sensitivity and specificity to endotoxins, the Limulus amebocyte lysate (LAL) assay has been employed to detect contaminating endotoxins in pharmaceuticals and parenteral drugs intended for human use. In addition, the LAL assay has been utilized clinically in the initial evaluation of meningitis, urinary tract infections, and urethritis-cervicitis syndromes.
The invention as described herein relates to a method and an apparatus for diagnostic assay or test of the existence of gonorrhea particularly in males. Although such tests have existed prior to the invention described herein, these tests, suffering under inadequacies as to their complexity, dependability, performance time, repeatability, have been expensive to prepare and of questionable accuracy. All of these tests have proved to require a substantial amount of time before accurate diagnosis can be made. With the method and apparatus described herein, many of these problems are overcome.
Both from the type of diagnostic employed as well as the apparatus used, the speed and accuracy of the test is enhanced. The assay used is a LAL assay which has been shown to be particularly effective in the rapid presumptive diagnosis of gonococcal urethritis in males. Tests employing Limulus lysate are explained in detail in the British Journal of Venereal Diseases, Volume 55, No. 3, 1979, pages 179-182. The culture predictability of Neisseria gonorrhoeae (sensitivity) was 100%, specificity was 96.3%, and the overall accuracy was 98%, indicating that the test offers a significant advancement in the area of diagnostic tests for gonorrhea.
The tests described in the British Journal of Venereal Diseases mentioned above are rather cumbersome and do not lend themselves to clinical evaluation where simplicity and speed are essential. For example, the original tests employed plastic test tubes and pipettes for qualitative evaluation of the clinical specimen. The preparation of test tubes, dilution, and the use of pipettes are not practical when used by the physician.
Rather, it is preferable that the various elements required in the diagnostic assay tests be packaged in a self-contained kit including a specimen collection apparatus, dilution device, and lysate assay material, such that the physician can use the assay immediately or a short period of time thereafter such as one-half hour. Diluting the specimen must be accomplished without contamination since the Limulus test detects picogram quantities of endotoxin. The device must also be simple and easy to use.
The invention relates to a method and apparatus which achieves the desired objectives discussed above in a unique manner which is efficient, simple, accurate and repeatable. More specifically, the apparatus relates to a kit which includes all the needed elements, including the solutions required for diluting a specimen and diagnosing the specimen after proper dilution, all without contamination. The elements of the kit include a sterile, non-pyrogenic syringe, such as a tuberculin syringe, with a cap, a sterile non-pyrogenic dilution container with a frangible closure, and a sterile non-pyrogenic needle with sheath or protector and a single test vial of Limulus lysate--lyophilized.
The syringe is initially used without the needle to take the specimen under partial vacuum when the plunger is pulled. Other items evaluated for the quantitative collection of urethral exudate have included microdiluter probes and capillary tubes. These have not been successful because the exudate is rather viscous requiring some kind of negative pressure to extract necessary amount of specimen for use in the diagnostic test. In addition, the pointed capillary tubes have the potential of harming the patient. Therefore, the tuberculin syringe without a needle has proved to be a preferable device to collect the urethral discharge under sufficient negative pressure and without potential injury to the patient while also maintaining good visibility during the collection operation.
The dilution step in the process must be accurate, quick, simple and inexpensive while avoiding the potential of contamination with respect to the specimen or creating a potential bio-hazard to the user. It is desirable that in diluting the specimen the system be sealed and remain sealed during mixing and dilution until the specimen is added to the lysate. The dilution device used is a container of diluent having a frangible closure. The device is specifically configured to cooperate with the syringe such that when the syringe is inserted into the dilution device, after the frangible closure is broken, a seal will be maintained between the two elements to permit dilution in a closed system and in a single step. The diluent used in the test herein is sterile, pyrogen-free water (H.sub.2 O), isotonic, pyrogen free solutions such as aqueous NaCl, etc.
The needle is then affixed to the syringe with the diluted specimen therein, and subsequently the needle, affixed to the syringe, is inserted into a vial septum and a measured portion of the diluted specimen is delivered to the LAL contained in the vial by pressing the plunger of the syringe.
In other embodiments the needle configuration is such that the needle may be placed on the syringe prior to dilution and enables the frangible closure to be broken by the needle as affixed to the syringe. This eliminates a separate element which may otherwise be required for breaking the frangible closure. In any event after the diluted specimen has been delivered to the vial, the specimen and LAL mixture after incubation for about 30 minutes is viewed to determine under observation whether gelation has occurred. Where there is gelation, the test presumptively indicates the presence of N.gonorrhoeae.
In taking specimen for the presumptive test in conjunction with diagnosis of gonococcal and nongonococcal cervicitis in females, there are additional problems in actually obtaining the specimen from the cervical os. In the laboratory the samples can be collected using a pipette with a gentle aspiration and subsequently diluting the sample in pyrogen-free water. Although these methods are acceptable and produce a high degree of sensitivity and specificity (Spagna et al., American Journal of Obstetrics and Gynecology, Vol. 137, No. 5, Pages 595-599, 1980), this approach is not practical for the practicing physician. Problems accompanying the use of the pipette occur because of the location of the cervix and the varying consistency and tenacity of the exudate. In addition, a series of dilution steps required would increase the possibility of error and of contamination to the sample and to the person actually employing the apparatus for taking the sample since the sample may contain biohazards. The invention is directed to overcoming these problems and arriving at a self-contained collection-dilution device which can be used by the physician at the patient's side. The device, as with the kit for the male presumptive diagnosis, includes the materials for collection of the specimen from the endo-cervix, for dilution of the specimen and for the subsequent LAL assay using a single-test lysate vial.
An aspiration step may be employed for the female where the specimen is aspirated in excess of the quantity needed and is subsequently delivered to or transferred to a syringe assembly which is then affixed to the diluent reservoir. This approach however requires several steps, and aspiration of the exudate from the endocervix, although possible, should be avoided, because it requires additional manipulation, is prone to errors and contamination with exogenous endotoxins. Acceptance of such a meticulous method by practicing physicians is difficult.
It is desirable to arrive at a method which avoids the problems above and is one which the physician is familiar with and can use quickly and accurately for rapid dilution and ease of operation. One procedure which is familiar to physicians in collecting endocervical samples and involves little or no risk to the patient is the use of a cotton tip swab. The swab is inserted into the cervical os about one to two centimeters. Although cotton is preferred since it is wettable, pyrogen-free polyester swabs or other devices might work, but the absorbent cotton should collect more endotoxins. Once the sample is obtained on the cotton tip the sample is diluted and subsequently delivered to LAL assay for determining the presumptive presence of N.gonorrhoeae.
The above discussion of the background of the invention and elements of the apparatus and the method for taking the diagnostic assay are only general in nature. A more detailed discussion of the elements of the invention as well as the details of its operation in connection with the method are described in a description of the preferred embodiment hereafter.